Understanding mechanisms of resistance to anaplastic lymphoma kinase (ALK) inhibitors and devising alternative treatment strategies to prolong patient survival are major research challenges with relevance to multiple cancer types. Anaplastic large-cell lymphoma (ALCL) is an ALK-driven tumor affecting primarily children and young adults. Optimum strategies for use of ALK-kinase inhibitors in ALCL are undefined. Our long-term goal is to develop therapies for ALK-driven cancers that prolong therapeutic control and overcome resistance to ALK-kinase inhibitors. The overall objective of this application is to establish a preclinical proof-of-principal for intermittent dosing of ALK-kinase inhibitors by exploiting the toxic effects of ALK kinase overdose we have observed in our preliminary data. Our central hypothesis is that while ALK up-regulation promotes resistance to kinase inhibitors, it makes cells dependent on continued inhibitor exposure to avoid ALK overdose, creating a system amenable to intermittent dosing. The rationale that underlies our proposal is that it will permit clinical evaluation of intermittent dosing of kK inhibitors to prolong patient survival in appropriate cases. We will test our hypothesis by pursuing three specific aims: 1) Determine how ALK up-regulation promotes resistance to ALK kinase inhibitors; 2) Identify the mechanism by which ALK overdose promotes cell death; and 3) Assess preclinically kinase-inhibitor intermittent dosing in vivo against tumors that employ ALK up-regulation as a resistance mechanism. Our approach in aim 1 examines the contribution of signaling pathways downstream from ALK activity through gene-expression profiling and gain- and loss-of-function experiments in ALCL model systems. Importantly, we have established systems in which fusion-ALK up-regulation promotes resistance in isolation from other resistance mechanisms. In Aim 2, we functionally interrogate the signaling that causes death due to ALK overdose. We also employ unbiased temporal assessments of gene expression as cells lose viability to gain a mechanistic understanding of the contribution from oncogene-induced senescence, negative feed- back signaling, and other mechanisms. In Aim 3, we undertake comprehensive in vivo studies of xenografted ALK+ ALCL cells and employ an NPM-ALK transgenic mouse model to confirm inhibitor-dependence of resistant cells, test for regression of tumors that develop resistance during in vivo selection, and compare intermittent vs. continuous dosing as up-front strategies. The contribution of the proposed research is both to identify the key pro-survival targets of ALK kinase up-regulation that promote resistance in the presence of inhibitor and to determine why ALK overdose results in cell death when inhibitor is withdrawn. This contribution will be significant because it will define the mechanisms of a major resistance marker thwarting the clinical success of ALK inhibitors while simultaneously establishing a new treatment paradigm to overcome it. This work is innovative, in our opinion, because our approach permits us not only to exploit oncogene overdose to therapeutic benefit in ALK-driven tumors for the first time, but to define the mechanisms by which it works.